The ability to detect and characterize low levels of microorganisms in biological samples is valuable for many applications including diagnosing and treating infections in both humans and animals, infectious disease research, detecting food contamination and identifying the causative organisms, monitoring product quality during food processing, monitoring environmental quality and so on.
Culture is often used to facilitate the detection and characterization of microorganisms in biological samples. The samples arc incubated in an atmosphere and at a temperature that is conducive to the growth of microorganisms, possibly with the addition of nutrient media to sample. Under these conditions, the microorganisms will multiply and can reach high concentrations. After growth to a sufficient concentration is achieved, a variety of methods can be used for the detection and characterization of the microorganisms. These methods include staining, fluorescence-in-situ-hybridization (FISH), polymerase-chain-reaction (PCR) and matrix-assisted-laser-desorption-ionization (MALDI) mass spectrometry. The drawback to culture is that it is slow, typically proceeding over many hours. Direct, i.e. non-culture, methods would therefore be preferred in those cases where rapid detection and characterization is important.
A range of bioanalytical methods rely on the lysis of cells for the release of intracellular components. Such components include organelles such as mitochondria, lysosomes, and endoplasmic reticulum, molecular assemblies such as microtubules and ribosomes and molecules such as proteins, carbohydrates and nucleic acids. Following lysis, the intracellular components can be subjected to analysis by for example electrophoresis, chromatography, mass spectrometry or optical spectroscopy. Likewise, molecular methods such as PCR, microarray analysis and sequencing rely on cell lysis for the release of intracellular DNA and RNA for amplification and other kinds of processing. To meet these needs, various cell lysis methods have been developed. Such methods include osmotic, chemical, mechanical (e.g. grinding with beads), hydrodynamic (e.g. pressure cell) and acoustic (i.e. sonication with ultrasound).
Ultrasound (acoustic waves beyond the audible range) has been used to lyse cells to release contents for molecular analysis often in conjunction with beads. See Seiter, J. A. and Jay, J. M. 19805. U.S. Pat. No. 5,374,522 (Murphy et al.) describes the use of an ultrasonic bath to disrupt cells such as Mycobacterium tuberculosis in a sample to which beads of glass or other materials in the range of 50 microns to 1 mm have been added. Such disruption released RNA and DNA into solution for hybridization with genetic probes. In U.S. Pat. No. 6,431,476, Taylor et al. teach a method for disrupting cells or viruses in a chamber with an ultrasonic transducer. Chandler et al. (U.S. Pat. No. 6,506,584) teach treating liquid with ultrasound in a flow-through device. The treatment can include cell lysis. U.S. Pat. No. 6,686,195 (Colin et al.) teaches lysing cells in a tube brought into direct contact with a shaped sonotrode. In U.S. Pat. No. 6,881,541 Petersen et al. teach a method for extracting nucleic acid from a sample using ultrasound. In U.S. Pat. No. 6,887,693 McMillan et al. teach a method for lysing components of a fluid sample that have been captured on a solid support. In U.S. Pat. No. 6,893,879, Petersen et al. teach a method for extracting an analyte from a fluid sample. U.S. Pat. No. 6,939,696 (Llorin et al.) teaches disrupting microorganisms in a sonicator at high pH in a lube without beads. In these references, the goal is to disrupt or lyse cells, whether mammalian or bacterial, to release the cell content for analysis. Belgrader et al. (U.S. Pat. No. 7,541,166) describe an apparatus that allows a sample or parts of a sample to be moved into a sonication chamber multiple times, allowing differing sonication levels to be applied to more and less sensitive cells such as epithelial and sperm cells releasing their DNA for analysis at different times.
In analyzing cell-containing biological samples, it is sometimes advantageous to lyse a subpopulation of the cells present in the sample. For example, when it is desired to perform a differential analysis of the white blood cells in blood using a Coulter counter, it is convenient to lyse the red blood cells while leaving the white blood cells intact. Various lysis solutions have been developed to achieve this result. See for example, U.S. Pat. No. 3,874,852 (Hamill), U.S. Pat. No. 4,185,964 (Lancaster), U.S. Pat. No. 4,521,518 (Carter et al.), U.S. Pat. No. 5,284,940 (Lin et al.), and U.S. Pat. No. 5,958,781 (Wong et al.). It is worth noting that red blood cells lyse fairly readily compared to the white blood cells and selective red blood cell lysis can be accomplished simply with osmotic shock. Agents that selectively lyse bacteria but not mammalian cells have potential utility in combating infections. Oren and Shay studied melittin diastereomers that lyse bacteria but not mammalian cells'. Selective lysis can be useful for biological research. Grifantini and coworkers were able to isolate adherent bacteria co-cultured with epithelial cells for gene expression studies by selectively lysing the epithelial cells with saponin.4 
Direct assays for the detection of microorganisms in biological fluids are often hampered by the presence of endogenous cells in high numbers. In general, such assays can be simplified if a method for selectively removing the endogenous cells were available. Zierdt and his colleagues published a lysis method in 19771. This method uses a mild detergent solution containing an enzyme mixture (Rhozyme prepared from Aspergillus oryzae cultures). In a subsequent paper2, Zierdt refined the solution by substituting the less toxic detergent Tween 20 for the Triton X-100 used in the original protocol. The Zierdt method is able to process a suitable volume of blood, 1 mL for example, in 1 hour, yielding a clear, red solution that can be filtered through a 0.6 micron track-etch filter 8 mm in diameter in approximately 3 minutes using a pressure differential of 2.5 psi. A key advantage of the Zierdt method is that the product is filterable through filters with pores small enough to retain microorganisms. Following filtration, the filter can be placed on a nutrient plate under suitable conditions, allowing colonies to grow from individual cells. The colonies can then be counted and further analyzed for the identity and antibiotic susceptibility of the organisms. Alternatively, FISH or other fluorescent labeling methods can be applied to the cells and fluorescence microscopy used to directly visualize the cells on the filter. This offers the possibility of rapid detection and identification of microorganisms in a range of complex samples including blood and other clinical specimens. Hence, a method that is able to selectively lyse mammalian cells faster than the Zierdt method would be advantageous.
In addition to the presence of cells, other constituents of biological samples can also hamper the detection of microorganisms. For example, bronchial samples are often highly viscous due to the presence of phlegm and other lung exudates. Urine specimens may contain significant amounts of protein as well as cells and mucus. These materials impede the detection of microorganisms by microscopic methods. Various reagents are used to overcome the obstacles to detection posed by these sample constituents. For example, N-acetyl-L-cysteine (NALC), combined with sodium citrate is a digestant that breaks up mucus in sputum and other bronchial samples. The sodium citrate stabilizes the NALC by binding heavy metal ions that may be present. Such reagents have proven to be useful, but their action is often slow and their effectiveness limited.
It is therefore an object of the present invention to provide a method for the rapid and efficient lysis of mammalian cells in biological samples while leaving microorganisms (bacteria and fungi) in the sample substantially intact.
It as a further object of the invention to provide a method for treating viscous, cell and protein containing biological samples to render them liquid and freely flowing without disrupting microorganisms that may be present.
It is a further object of the invention to provide a method for making highly cellular and/or viscous biological samples filterable through small pore size filters in order to retain and concentrate microorganisms on the filter for further analysis.